AR-related Assays (Androgen)

luciferase reporter constructs validated for androgen receptor

The LightSwitch™ Validated Androgen Receptor (AR) Pathway Collection is a comprehensive set of sequence-verified, transfection-ready, promoter reporter constructs that can be used to accurately quantify transcriptional activation and repression in response to induction of pathways regulated by the androgen receptor. These constructs are a subset of the 18,000-member LightSwitch Promoter Reporter GoClone™ Collection that were selected based on sequence motif analysis, published information and in-house analysis of each promoter's activity* using our LightSwitch Luciferase Assay.

The AR Biomarker Set contains 8 of the highest responding promoters under our chosen test conditions, as well as the promoters of 2 housekeeping genes (Figure 1). The AR Profiling Plate contains 88 promoter reporter constructs that showed significant activation or repression in response to induction (See the AR Profiling Plate tab below for data and a list of constructs).

Figure 1: Induction ratios for each member of the AR Biomarker Set.

HT1080 human fibrosarcoma cells were co-transfected with the promoter reporter constructs shown and an AR cDNA expression plasmid, then treated with 10 nM methyltrienolone (R1881) or vehicle only. After 24 hours, each sample was assayed using the LightSwitch Luciferase Assay Kit. The graph shows the ratio of treated/untreated signal on a log scale; error bars represent one standard deviation. “NONE” = No gene annotation. Please see the heatmap under the Profiling Plate tab below for relative promoter activities under untreated conditions.

*Experimental results and the absolute values of luciferase units may vary depending on cell line, treatment and protocols. All data shown is solely for use as a guide for selecting candidate promoters and panels for your particular experimental design.

Androgen Receptor (AR) Pathway

Nuclear receptors, ligand-activated transcription factors, are critical for understanding gene regulation and the genomic events that lead to development, differentiation and growth in an organism. Ligands for nuclear receptors (NRs) include a variety of small molecules such as steroids and fatty acids. Xenobiotics and various pharmaceutical agents also serve as NR ligands, illustrating the importance of NR-mediated toxicity screening.

Androgen receptors facilitate signaling of testosterone and other androgenic hormones in the cytoplasm; AR translocates into the nucleus upon hormone binding. AR serves as a DNA-binding activator of transcription of hormone-responsive genes. Androgen Receptor is involved in the development of both primary and secondary male sexual characteristics; abnormal expression of AR is linked to prostate cancer.

LightSwitch Promoter Reporter Assays.

To study a promoter's regulation, it is cloned into a LightSwitch Reporter vector upstream of the RenSP luciferase gene and then transfected into mammalian cells. The cells are stimulated, if required, to induce transcription of RenSP luciferase from the cloned promoter. Promoter activity is then quantified using a LightSwitch Luciferase Assay Kit and a luminometer to measure the amount of luminescence produced.

Cost-effective – Efficiently screen for activation and/or repression using a multitude of conditions.

LightSwitch™ Androgen Receptor (AR) Pathway Profiling Plate

The AR Profiling Plate is a subset of promoter reporter constructs chosen from the LightSwitch Promoter Reporter GoClone™ Collection. Promoters that were expected to be up- or down-regulated in the androgen receptor pathway were chosen based on motif analysis and published information. In-house luciferase testing was then performed to select those promoter reporter constructs that were activated or repressed in response to our induction method, which was to treat HT1080 cells with 10 nM methyltrienolone (R1881) for 24 hours following co-transfection with a promoter-reporter construct and an AR cDNA expression plasmid.

AR Profiling Plate heatmap

The heatmap at right shows the untreated and induced activity for all constructs that are members of the pathway profiling plate. On the right side of the figure, the colors represent log2 transformed ratios of treated/untreated activity according to the color scale shown at the base of the heatmap. Black indicates no change; the intensity of red or blue color indicates levels of induction or repression, respectively. On the left side of the figure, the intensity of yellow color depicts the activity of each promoter in the unstimulated (untreated) condition. All untreated signals are relative to the highest and lowest values within the individual experiment. (“R1881” = HT1080 fibrosarcoma cells treated with 10 nM methyltrienolone (R1881) for 24 hours following co-transfection with a promoter-reporter construct and an AR cDNA expression plasmid.)

AR Profiling Plate contents

The AR Profiling Plate includes 88 promoter reporter constructs (including the 8 top-responding constructs that comprise the AR Biomarker Set, as well as 8 housekeeping / random promoters that are unlikely to respond to AR induction conditions). The controls can be used to correct for non-specific changes in overall signal in response to the stimulus. For example, some compounds used for induction are very toxic and result in a significant decrease in overall luciferase signal across the entire panel of constructs in a non-promoter-specific manner. Also included in the plate are 8 transfection controls that are used to determine the conditions that yield maximum transfection efficiency and reproducibility.

Ordering

If you would like to order 5 or fewer members of the AR Profiling Plate, you can use the Product IDs found in the file for the plate map above to order directly on Active Motif's SwitchGear Genomics website. If you wish to order the entire plate (or a minimum of 5 or more members), click here to send us an e-mail detailing which constructs you would like, and what amount (5 µg, 10 µg, 1 mg, etc.). We will send you a quote based on your request.

Click image to enlarge.

The AR Biomarker Set contains 5 µg aliquots each of 8 promoter reporter constructs and 2 housekeeping promoter constructs (NO_ANNOTATION & NUB1) for use as controls. Click on the links below for clone information.